Kirchner et al. Medicine (2016) 8:92 DOI 10.1186/s13073-016-0343-7

REVIEW Open Access Recent advances in malaria and epigenomics Sebastian Kirchner, B. Joanne Power and Andrew P. Waters*

Abstract Malaria continues to impose a significant disease burden on low- and middle-income countries in the tropics. However, revolutionary progress over the last 3 years in nucleic acid sequencing, reverse genetics, and post-genome analyses has generated step changes in our understanding of malaria parasite (Plasmodium spp.) biology and its interactions with its host and vector. Driven by the availability of vast amounts of genome sequence data from Plasmodium species strains, relevant human populations of different ethnicities, and mosquito vectors, researchers can consider any biological component of the malarial process in isolation or in the interactive setting that is infection. In particular, considerable progress has been made in the area of population genomics, with Plasmodium falciparum serving as a highly relevant model. Such studies have demonstrated that genome evolution under strong selective pressure can be detected. These data, combined with reverse genetics, have enabled the identification of the region of the P. falciparum genome that is under selective pressure and the confirmation of the functionality of the in the kelch13 gene that accompany resistance to the major frontline antimalarial, artemisinin. Furthermore, the central role of epigenetic regulation of gene expression and antigenic variation and developmental fate in P. falciparum is becoming ever clearer. This review summarizes recent exciting discoveries that genome technologies have enabled in malaria research and highlights some of their applications to healthcare. The knowledge gained will help to develop surveillance approaches for the emergence or spread of drug resistance and to identify new targets for the development of antimalarial drugs and perhaps vaccines.

Background to provide new avenues for therapeutic or prophylac- Malaria, caused by unicellular protozoan Plasmodium tic development based upon biological insights such spp. parasites, is an ancient disease and remains a as the identification of new drug targets and vaccine major threat to human health and wellbeing. Five candidates. species of Plasmodium are currently recognized as The landmark of the completion of the genome causing human malaria, of which the most lethal is sequence of a laboratory strain of Pf was achieved over P. falciparum (Pf). In 2015, the World Health a decade ago [2] (Fig. 1). This has since been accom- Organization estimated that the maximal annual bur- panied, thanks to plummeting costs and advances in den imposed by malaria, whilst decreasing, still stands next-generation sequencing (NGS) technologies, by the at 214 million (range 149–303 million) cases that whole-genome sequencing (WGS) of a wide range of result in 438,000 (range 236,000–623,000) deaths [1]. species representing all the major clades of the genus, Drug resistance to frontline antimalarials continues to although the of all known human infectious arise and spread, exacerbated by slow progress in the Plasmodium species remain to be sequenced [3]. How- introduction of alternatives. Properly efficacious vac- ever, the combination of NGS and WGS has enabled cines remain a hope, not a likelihood. Against this the development of innovative large-scale genomic background, genome-based research on malaria seeks studies, for example, for genomic epidemiology [4]. Such population genomics, fueled by collaborative * Correspondence: [email protected] consortia (for example, the Malaria Genomic Epidemi- Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, ology Network (MalariaGEN; http://www.malariagen.net), Immunity and Inflammation, College of Medical Veterinary & Life Sciences, University of Glasgow, Glasgow G12 8TA, UK have allowed the dynamics of global and local population

© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kirchner et al. Genome Medicine (2016) 8:92 Page 2 of 17

structures to be assessed and adaptive change in parasite The African Genome Variation Project recognizes the genomes to be monitored in response to threats, such significant diversity of ethnicities and, therefore, geno- as artemisinin (ART). This is especially true for single- typesand,throughWGS,imputation,andSNP nucleotide polymorphisms (SNPs), and while other mapping, seeks to build a database through which aspects of genome variation (such as indels and copy disease incidence and outcome can be reliably associ- number variation) might currently lag behind, the ated with haplotypes [7]. Already, such wider analyses gaps in the database are known and are firmly in the have confirmed SNP associations with five well-known traits, sights of researchers. including hemoglobinopathies and glucose-6-phosphate The template Plasmodium genomes have provided the dehydrogenase (G6PD) deficiency, but have refuted 22 substrate for the application of an explosion of other others that had been linked by smaller-scale studies [8]. This post-genome survey technologies that have been largely study also showed opposing effects of G6PD on different exclusively applied to Pf, such as transcriptomics, prote- fatal consequences of malaria infection, revealing a hitherto , , and , and that map the unsuspected complexity of associations. Ongoing analyses general and stage-specific characteristics of malaria para- have revealed new, although unsurprising, examples sites. These data are warehoused in expensive but critical of haplotypes of loci associated with protection from community web sites such as PlasmoDB (http://www.Plas severe malaria, such as the glycophorin locus on human modb.org). This in turn has been exploited by ever chromosome 4 [8, 9]. improving forward and reverse genetic capabilities to assign function to genes, steadily reducing the >60 % of Vector genomics genes of unknown function that were originally catalogued In Africa, malaria is mainly transmitted by female [2]. Advances that will be highlighted in this review Anopheles gambiae (Ag) mosquitoes. Approaches to include: the unraveling of the molecular mechanisms of understanding the role of Ag mosquito genomics in parasite resistance to ART; the functional identifica- malaria transmission have been similar to those of the tion of some of the -modifying enzymes that African Genome Variation Project. Thus, the Ag1000G write the epigenetic code (such as Pf histone deacety- project (https://www.malariagen.net/projects/ag1000g) lase 2 (PfHDA2)) and the that read it (such involves 35 working groups that have sampled Ag mosqui- as Pf heterochromatin 1 (PfHP1)) that, with tos from 13 malaria endemic countries and that aim to others (such as RNaseII), play a significant role in the establish the levels of Ag genome diversity, establish popu- regulation of antigenic variation and commitment to lation structures, and link these to the ecology of disease sexual development. transmission. The Anopheles vector genome is very Furthermore, the genomes of the host and of a grow- dynamic. Comparative vector genomics has revealed rapid ing number of mosquito vectors have been characterized gene gain and loss compared with Drosophila and signifi- in both increasing number and depth, permitting meta- cant intragenus diversity and mixing in genes involved analyses of these genomes in combination with Plasmo- in both insecticide resistance and antimalarial immun- dium infection. These studies have revealed important ity [10, 11]. The nature and extent of such diversity loci associated with resistance to the malaria parasite in precludes the application of classic GWAS approaches the host and vector, respectively [5, 6], and indicate the and a novel approach of phenotype-driven, pooled se- genomic hotspots in the genetic arms race that malaria quencing coupled with linkage mapping in carefully has stimulated. selected founder colonies has been used to map vec- We also review the recent advances in this very active tor phenotypes. This study recently revealed TOLL11 area of malaria genomics and control of gene expression as a gene that protects African mosquitoes against Pf and emphasize any benefits that these advances may infection [6]. have for the development of therapies and interventions (Table 1). Parasite genomics Full genome sequences are now available for many Human genomics strains of Pf [2], Plasmodium vivax [12], and Plasmo- The infrastructure required to effectively collect, collate, dium knowlesi [13] among the human infectious and analyze large genomes for epidemiological studies parasites. Primate and rodent infectious species that are (that is, genome-wide association studies (GWAS)) is so frequently used as model parasites have also been costly that it is best achieved in consortia. These can sequenced and include Plasmodium berghei (Pb), Plas- work at such a scale that analyses are powered to a de- modium cynomolgi, Plasmodium chabaudi and Plasmo- gree that GWAS findings become more certain and the dium yoelii [14]. Recently, the genomes of seven further global context of the effect of, for example, human genet- primate infectious species have become available, ics on susceptibility to malaria is more reliably resolved. demonstrating the close relationship between Pf and Kirchner et al. Genome Medicine (2016) 8:92 Page 3 of 17

Fig. 1 Major advances in omics-related fields. This figure highlights landmark studies providing key insights into parasite makeup, development, and pathogenesis (yellow boxes) as well as crucial technical advances (blue boxes) since the first Plasmodium genomes were published in 2002 [2, 5, 12, 13, 27, 29, 31, 39, 40, 42, 43, 48–50, 53, 54, 57, 66, 114, 115, 151, 153–178]. AID auxin-inducible degron, ART artemisinin, cKD conditional knockdown, CRISPR clustered regularly interspaced short palindromic repeats, DD destabilization domain, K13 kelch13, Pb P. berghei, Pf P. falciparum, TSS start site, TF , ZNF zinc finger nuclease chimpanzee infectious species [15]. The typical genes [16] and antisense transcription [17, 18] are Plasmodium genome consists of 14 linear chromo- being catalogued in Pf but this catalogue probably somes of aggregate size of approximately 22 mega- remainsincompleteasonlyblood-stageparasites bases encoding >5000 protein-coding genes. The core, have been seriously investigated in this regard and conserved genome of about 4800 such genes occupies ncRNAs remain largely of unknown significance. the central chromosomal regions whilst the multi- One key feature of Pf is its evolution in the face of gene families (at least some of which are associated human-imposed selection pressures in the form of drugs with antigenic variation) are largely distributed to the and potentially vaccines. Such pressure has consistently subtelomeric regions. Non-coding RNA (ncRNA) resulted in the emergence of drug-resistant parasites. Kirchner et al. Genome Medicine (2016) 8:92 Page 4 of 17

Table 1 Key advances from recent omics studies Research area Key findings Reference(s) Genome sequencing WGS and refinement of genome sequences of primate and rodent infectious Plasmodium [13, 154–157, 179] species Genome sequence of cryptic chimpanzee Plasmodium species [158] Gene expression regulation Discovery of AP2-G as master regulator of gametocytogenesis in Pf and Pb [49, 50] AP2-G2 functions as a transcription regulator driving gametocyte development [51] Functional analysis of Pf lifecycle dynamics using transcriptional profiling [180] Genome-wide TSS mapping in Pf [42] Acetylome analysis reveals post-translational PTM of ApiAP2 transcription factors [55] Genome-wide analysis of AP2-O target genes [48] Post-transcriptional/translational Genome-wide analysis reveals features of the Pf translatome [59, 66] control Analysis of the Pb maternal repressome [64] Epigenetic control SETvs functions as a regulator of var gene silencing in Pf [98] Involvement of HP1 in clonal variant gene expression and sexual development [29] HDA2 mediates var gene silencing and sexual commitment in Pf [27] BDP1 regulates expression of invasion-related genes [31] Profiling of histone PTMs in Pf [22] Gene silencing/editing First description of the use of ZFN and CRISPR-Cas9 genome editing in Plasmodium [115, 116, 140] Functional adaptation of an AID-based conditional knockdown system in Pb [159] Antibiotic resistance Analysis of KELCH13-propellermutations in Plasmodium field isolates confirms relation to arte- [115, 116] misinin resistance Identification of UPR pathway involvement in Plasmodium artemisinin resistance [123, 126] Genome structure First profiling of genome-wide chromosome interactions [39] Strong correlation between spatial genome structure and gene expression [37] ncRNAs PfRNaseII exonuclease is involved in group A var gene silencing [84] Genome-wide search revealed over 1000 novel ncRNAs in Pf [181] Strand-specific RNA sequencing reveals widespread transcription of antisense in Pf [16–18] Antisense lncRNAs regulate expression of cognate var genes in Pf [87] Virulence/transmission Vector transmission of P. chabaudi chabaudi impacts on parasite virulence [182] changes during early phase of mosquito infection [183] Development Global phosphoproteome analysis identified PKG as major intracellular Ca2+ level regulator [184] PKG regulates Plasmodium egress and evasion [185] Systematic subtractive bioinformatic analysis identified sex-specific stage V gametocyte pro- [186] tein marker Protein phosphatases are key regulators of Plasmodium development [187] Population genomics Identification of a novel malaria resistance locus through GWAS in African children [5] Genomic epidemiology study revels global KELCH13 distribution [160] Metablomics Lipid metabolism analysis in Pf [151] AID auxin-inducible degron, CRISPR clustered regularly interspaced short palindromic repeats, GWAS genome-wide association study, lncRNA long non-coding RNA, ncRNA non-coding RNA, Pb P. berghei, Pf P. falciparum, PTM post-translational modification, SNP single-nucleotide polymorphism, TSS transcription start site, UPR unfolded protein response, WGS whole-genome sequencing, ZFN zinc finger nuclease

There is a huge potential global reservoir of genome catalogue is described in detail by Manske and col- variation upon which selection may act. In an initial leagues [19]. Currently (27 July 2016), the MalariaGEN analysis of 227 parasite samples collected at six different database states that for the Pf Community Project, it locations in Africa, Asia, and Oceania, MalariaGEN, the has data on 3488 samples from 43 separate locations Oxford-based genomic epidemiology network, identified in 23 countries and the number of high-quality, fil- more than 86,000 exonic SNPs. This initial SNP tered exonic SNPs has increased to more than Kirchner et al. Genome Medicine (2016) 8:92 Page 5 of 17

900,000. All of this variation is diversity, which in turn can dysregulated by HDA2 knockdown are also known be selected for fitter and perhaps more deadly parasites. to be associated with HP1, a key epigenetic player Modern NGS and WGS have enabled comparative and binding to tri-methylated H3K9 (H3K9me3), linked to population genomics approaches that have been used to transcriptionally repressed . Strikingly, reveal important features of emerging parasite popula- conditional knockdown of PfHP1 recapitulated, to a tions, for example, in response to drugs. much greater extent, the phenotype observed in HDA2-knockdown mutants [29]. HP1 is believed to Parasite development and pathogenesis act as a recruitment platform for histone meth- Within their mammalian host and mosquito vector yltransferases (HKMTs), required for maintenance and Plasmodium parasites complete a remarkable lifecycle, spreading of H3K9me3 marks [30], which is consist- alternating between asexual and sexual replication ent with the reduction of H3K9me3 observed in HP1 (Fig. 2). Throughout the Plasmodium lifecycle, regula- knockdown cells [29]. In addition, bromodomain pro- tion of gene expression is orchestrated by a variety of tein 1 (BDP1) was found to bind to H3K9ac and mechanisms, including epigenetic, transcriptional, post- H3K14ac marks within transcription start sites (TSSs) transcriptional, and translational control of gene expres- in Pf, among them predominantly invasion-related sion. Owing to the absence of most canonical eukaryotic genes (Fig. 3a), and BDP1-knockdown parasites con- transcription factors in the Plasmodium genome [2], sistently failed to invade new erythrocytes. BDP1 also epigenetic control has long been recognized to play an appears to act as a recruitment platform for other important role in gene expression regulation. effector proteins such as BDP2 and members of the lies at the very heart of gene expression, apicomplexan AP2 (ApiAP2) transcription factor regulating access of the transcriptional machinery to family [31]. chromatin [20] via (1) post-translational modifications In addition to histone PTMs, organization (PTMs) of , (2) nucleosome occupancy, and (3) plays a critical role in gene expression regulation in global chromatin architecture. In the past decade, Plasmodium. In general, heterochromatin is substantially various histone PTMs have been identified throughout enriched in compared with euchromatin the Plasmodium lifecycle (reviewed in [21]) and the [32] and active promoters and intergenic regions in Pf existing catalog of modifications in Pf was recently show markedly reduced nucleosome occupancy [33]. In extended to 232 distinct PTMs, 88 unique to Plasmo- addition, common transcript features such as TSSs, dium [22]. The majority of detected PTMs show transcription termination sites, and splice donor/ dynamic changes across the intra-erythrocytic develop- acceptor sites show clearly distinguishable nucleosome ment cycle (IDC), likely mirroring changes within chro- positioning in Pf [34], but previously described dy- matin organization linked to its transcriptional status. namic changes in nucleosome positioning [32] ap- and of N-terminal histone tails peared to be mostly restricted to TSSs during the are by far the most studied regulatory PTMs, linked IDC [34]. Uniquely in Plasmodium spp., canonical either to a transcriptionally active chromatin structure histones in intergenic regions are replaced by histone (that is, euchromatin) or to transcriptionally inert het- variant H2A.Z [28], which, in concert with the erochromatin. In Pf, various genes encoding putative apicomplexan-specific H2B.Z, establishes a H2A.Z/H2B.Z epigenetic modulators (that is, proteins catalyzing either double-variant nucleosome subtype enriched at AT-rich the addition or removal of histone PTM marks) have regions and correlates with open chromatin and been identified [23], but only a few have been subjected active gene transcription [35]. to more detailed investigation [24, 25]. Many of the Within the confined space of the nucleus, chromo- histone modifiers are essential for Plasmodium develop- somes are tightly packed into a three-dimensional ment, making them a promising target for antimalarial structure. This three-dimensional architecture allows drugs [26]. In Pf, conditional knockdown of HDA2, a interaction between otherwise distant chromatin regions histone lysine deacetylase (HDAC) catalyzing the re- possessing regulatory function and facilitates contacts moval of acetyl groups from acetylated histone 3 ly- with other nuclear sub-compartments such as the sine 9 (H3K9ac), resulted in elevated H3K9ac levels nucleolus and the nuclear envelope [36]. Until recently, in previously defined heterochromatin regions [27]. knowledge of the chromosome architecture and chroma- H3K9ac is an epigenetic mark associated with tran- tin interactions in Plasmodium was mostly restricted to scriptionally active euchromatin [28] and HDA2 de- single genomic loci based on fluorescence in situ pletion resulted in the transcriptional activation of hybridization experiments [37]. However, recent advances genes located in heterochromatin regions, leading to in deep-sequencing technologies [38] have for the first impaired asexual growth and an increased gametocyte time enabled the genome-wide profiling of chromosome conversion [27]. Interestingly, genes found to be interactions at kilobase resolution in Plasmodium [37, 39]. Kirchner et al. Genome Medicine (2016) 8:92 Page 6 of 17

Mosquito blood meal

Salivary Liver gland Hepatocyte

Schizont

Sporozoites

Merozoites

Oocyst

Merozoites Ookinete RBC Midgut Blood stage Zygote

Ring

Schizont Gametes

Mosquito Trophozoite blood meal

IDC Gametocytes Mosquito vector Mammalian host

Fig. 2 Plasmodium life cycle. After a mosquito bite, malaria parasites are deposited into the host’s skin and within minutes are carried via the bloodstream into the liver, where through asexual proliferation within the hepatocytes tens of thousands of merozoites are produced. Following hepatocyte rupture, merozoites are released into the bloodstream where they can invade the host’s red blood cells (RBC), leading to the initiation of the intra-erythrocytic development cycle (IDC). During the IDC (lasting about 48–72 h in human and about 24 h in rodent malaria parasites), Plasmodium parasites multiply asexually through the completion of several morphologically distinct stages within the RBCs. After RBC invasion, malaria parasites develop via the ring and trophozoite stage into schizonts, each containing a species-specific number of merozoites (typically 10–30). Upon schizont rupture, merozoites are released into the bloodstream, where they can invade new RBCs and initiate a new IDC. However, a small fraction of ring-stage parasites sporadically differentiate into male or female gametocytes, which are responsible for initiating transmission back to the mosquito. Through another mosquito blood meal gametocytes are taken up into the mosquito midgut where they are activated and form male (eight per gametocyte) and female (one) gametes. Following fertilization, the zygote undergoes (and therefore true sexual recombination) and develops into a motile, tetraploid ookinete that traverses the midgut and forms an oocyst. Via another round of asexual proliferation inside the oocyst several thousands of new haploid sporozoites are generated that, upon their release, colonize the mosquito salivary glands, poised to initiate a new infection of another mammalian host

In contrast to other eukaryotic organisms, the Pf nucleus Transcription itself is initiated through binding of the appears to lack clearly defined chromosome territories transcriptional machinery to promoter regions in the and chromatin interactions are mainly restricted to intra- nucleus, resulting in the synthesis of pre-mRNA mole- chromosomal contacts showing a clear distance-related cules, which, following extensive processing and nuclear dependency [37, 39]. Inter-chromosomal interactions are export, leads to the accumulation of mature mRNAs in mostly absent in Pf and restricted to centromeres, the parasite cytosol [41]. A recent study found evidence telomeres, ribosomal DNA (rDNA) loci, and internal as for stage-specific transcription initiation from distinct well as subtelomeric-localized var genes (further discussed TSSs of otherwise identical transcriptional units, giving in the next section). This observed clustering appears to rise to developmentally regulated mRNA isoforms [42]. coincide with transcriptional activity of each cluster. While most canonical eukaryotic transcription factors Interestingly, using three-dimensional chromatin model- are absent from the Plasmodium genome [2], the ing, the highly transcribed rDNA genes were proposed to ApiAP2 family of DNA-binding proteins comprises by be localized to the nuclear periphery, which was far the largest group of transcription factors in malaria previously mainly associated with transcriptionally si- parasites [43]. A collection of ApiAP2 proteins is lenced heterochromatin [40], indicative of perinuclear expressed throughout all stages of the IDC [44], while transcriptionally active compartments [37]. other ApiAP2 proteins are expressed outside the IDC Kirchner et al. Genome Medicine (2016) 8:92 Page 7 of 17

Fig. 3 Malaria parasite genomic components involved in pathogenesis. a The expression of invasion-related genes is regulated through epigenetic and post-transcriptional mechanisms. Bromodomain protein 2 (BDP2) binds to H3K9ac marks within the promoter region of genes associated with red blood cell (RBC) invasion (as well as other gene families not depicted here [31]), enabling their transcription. This is likely achieved through the recruitment of BDP1 and transcription factors (TFs) of the ApiAP2 family. Following transcription during the trophozoite stage, mRNAs encoding invasion-related proteins are bound by ALBA1 functioning as translation repressor. After progression to the schizont stage, ALBA1 is released, allowing the timely synthesis of proteins required for merozoite invasion of RBCs. b Experimental findings either directly from studies on ap2-g or from epigenetically regulated var genes are suggestive of an epigenetically controlled mechanism regulating ap2-g transcription. In sexually committed parasites, ap2-g is characterized by H3K4me2/3 and H3K9ac histone marks and most likely contains histone variants H2A.Z and H2B.Z located in its promoter region. BDPs are believed to bind to H3K9ac, facilitating ap2-g transcription. ApiAP2-G drives expression of genes required for sexual development through binding to a 6/8-mer upstream DNA motif. ap2-g expression itself is believed to be multiplied through an autoregulatory feedback loop where ApiAP2-G binds to its own promoter that also contains ApiAP2-G motifs. In asexual blood-stage parasites, ap2-g is transcriptionally silenced by heterochromatin protein 1 (HP1) binding to H3K9me3 histone marks (located in repressive loci in the nuclear periphery). 2 (HDA2) catalyzes the removal of H3K9ac from active ap2-g, facilitating ap2-g silencing. c Monoallelic expression of one of the approximately 60 members of the erythrocyte membrane protein 1 (EMP1)-encoding var genes is regulated through epigenetic silencing of all but one var gene copy. The active var is marked by euchromatin post-translational modifications H3K4me2/3 and H3K9ac and histone variants H2A.Z/ H2B.Z located in its promoter region, as well as H3K36me3 covering the whole var gene body but absent from the promoter region. Transcription of noncoding RNAs associated with the active var gene is facilitated by upstream as well as intronic promoters. All other silenced var genes cluster into perinuclear repressive loci and are characterized by HP1 binding to H3K9me3 marks. var gene silencing also involves SET2/vs-dependent placing of H3K36me3 histone marks in promoter regions and is marked by the absence of non-coding RNAs, likely safeguarded through RNaseII exonuclease activity. In addition, other modulators such as HDA2, SET10, and SIR2A/B are likely involved in epigenetic var gene regulation. d Mutations in kelch13 (K13) were found to be the major contributors to artemisinin (ART) resistance identified in drug-resistant parasites in the laboratory as well as in field isolates. kelch13 mutations appear to arise in a complex array of background mutations (that is, mutations in genes encoding ferredoxin (FD), apicomplast ribosomal protein S10 (ARPS10), multidrug resistance protein 2 (MDR2), and chloroquine resistance transporter (CRT)), not yet detected in African parasites. In addition, elevated phosphatidylinositol-3-kinase (PI3K) levels have been observed in ART-resistant parasites and PI3K signaling has been implicated to impact on the unfolded protein response observed in ART-resistant parasites. H2A.Z/H2B.Z, orange/yellow-paired quarter circles; H3K4me2/3, light green circles;H3K9ac,dark green circles; H3K9me3, red circles; H3K36me3, blue circles; canonical nucleosomes, grey globes; ApiAP2-G binding motif; light blue line;ncRNAs,wobbly red lines; mRNAs, wobbly black lines. AP2n other TFs belonging to the ApiAP2 DNA binding protein family, ncRNA non-coding RNA, TFs transcription factors Kirchner et al. Genome Medicine (2016) 8:92 Page 8 of 17

[45–47]. ApiAP2s appear to be among the main drivers genome-wide analysis of the Plasmodium translatome. of developmental progress throughout most Plasmodium Throughout the IDC, transcription and translation are lifecycle stages and their disruption abolishes or greatly tightly coupled and only 8 % (approximately 300 tran- reduces parasite development [45, 46]. They bind in a scripts) of the transcriptome was found to be transla- sequence-specific fashion to motifs generally distributed tionally regulated [59]. These genes were found to be upstream of open reading frames (ORFs) and individual involved in merozoite egress and invasion, and while AP2s may have widespread influence; PfAP2-O has been transcript level peaked during the late stages of the IDC, shown to bind upstream of >500 genes (roughly 10 % of maximal translation was observed during the early ring the parasite ORFs), potentially influencing a wide range stage. This observation resembles a general feature of of cellular activities [48]. gene expression in Plasmodium, whereby for a set of Through forward genetic screens and comparative genes transcription and translation are uncoupled and genomics, ApiAP2-G was discovered to function as a mRNA translation occurs during a later developmental conserved master regulator of sexual commitment in Pf time point when compared with maximal transcriptional and Pb. ApiAP2-G binds to a conserved 6/8-mer nucleo- activity, most notably in female gametocytes [46, 60–64]. tide motif enriched upstream of gametocyte-specific This is especially true for genes required for develop- genes and ap2-g itself, leading to an autoregulatory feed- mental progression and provides the parasite with the back loop [49, 50] (Fig. 3b). ApiAP2-G2, another capability of rapid and timely protein synthesis without ApiAP2 family member, acts downstream of ApiAP2-G the need for preceding de novo mRNA synthesis. during sexual development, functioning as a transcrip- Recently, PfALBA1, a member of the DNA/RNA- tional repressor blocking expression of genes required binding Alba protein family, was postulated to act as for asexual development and influencing gametocyte sex master regulator during the Pf IDC, controlling trans- ratios [50, 51]. During the asexual IDC, ap2-g displays lation of invasion-related transcripts (Fig. 3a) as well characteristics of epigenetically silenced heterochroma- as regulating mRNA homeostasis of approximately tin, such as H3K9me3 marks, binding to HP1 and 100 transcripts in blood-stage parasites [65]. In localization to the nuclear periphery (reviewed in [52]) contrast to findings by Caro and colleagues [59], an (Fig. 3b). However, the previously mentioned knock- earlier study using polysome profiling found a dis- downs of both PfHDA2 and HP1 resulted in increased crepancy between steady-state mRNA level and gametocyte conversion, likely as a direct consequence of polysome-associated mRNAs among 30 % of genes the loss of H3K9me3 marks and H3K9 hyperacetylation (1280 transcripts) during the Pf IDC, indicative of leading to ap2-g transcriptional activation [27, 29]. This translationally controlled gene expression [66]. Add- opens the possibility of a bet-hedging mechanism for itionally, the results of this study, as well the findings sexual commitment in Plasmodium, regulating sto- of others, suggest upstream ORF translation and stop chastic, low-level activation of ap2-g sensitive to en- codon read-through in Pf [67–69], but the genome- vironmental stimuli, as has been shown for several wide extent of such mechanisms in Plasmodium spp. blood-stage-expressed genes [52, 53]. PTMs such as remains controversial [59]. Hence, expansion of these lysine acetylation are not restricted to histones and a studies to other parasite life stages, such as the recent study has demonstrated that the “acetylome” gametocyte, where translational control is firmly impacts >1000 proteins and intriguingly is highly established, would surely give further insights into the enriched in the ApiAP2 transcription factor family extent of translation regulation in Plasmodium. [54, 55], although the functional consequences of In addition to canonical protein-coding mRNAs, a vast these PTMs have yet to be established. number of genes encoding different ncRNAs have been Following their synthesis, eukaryotic mRNAs are proc- identified within the Plasmodium genome in recent essed and are finally translated by the ribosomal machin- years, which are believed to exert a variety of regulatory ery. Translation has long been a focus of malaria functions (reviewed in [70]). Circular RNAs (circRNAs) research, not only because it represents a promising tar- are among the newest members of the still expanding get for antimalarial drugs but also for its potential regu- catalogue of existing ncRNAs in Plasmodium [17]. Host latory features [56]. The lack of correlation between microRNAs (miRNAs) have been shown to regulate transcript and protein level observed throughout the parasite translation [71], and circRNAs therefore might Plasmodium lifecycle has fueled researchers’ interest in act as sponge for host miRNAs, a mechanism described post-transcriptional and translational control for decades in other organisms [72]. Recent studies have especially [57]. Many features of post-transcriptional/translational increased our knowledge of the role of ncRNAs in var control in malaria parasites are similar to the mecha- gene regulation (discussed in the next section) but, nisms found in other [41]. However, the nevertheless, the biological role of the vast majority of advent of ribosome profiling [58] has enabled in-depth these ncRNA species remains unclear. Kirchner et al. Genome Medicine (2016) 8:92 Page 9 of 17

Immune evasion heterochromatin formation [40, 89, 90, 92]. Conditional In their attempts to occupy a diverse range of host envi- disruption of one of these essential proteins, HP1, dis- ronments, protozoan parasites of the Plasmodium genus rupts singular var gene expression and dysregulates anti- have evolved a plethora of molecular mechanisms to genic variation [29]. In addition, conditional knockdown evade the host adaptive immune response. The host of PfHDA2 has been shown to result in a dramatic loss immune response to Plasmodium infection is dependent of monoallelic var gene expression [27]. This implicated upon both host and parasite genomics and the develop- PfHDA2 as an upstream regulator of HP1 binding as it mental stage and phenotype of the invading parasite facilitates the establishment of the H3K9me3 mark. The [73–75]. In the best-studied example in Plasmodium, indispensable role of the dynamic histone lysine methy- virulence of Pf is attributed largely to monoallelic lation of Plasmodium chromatin by histone lysine expression of just one of approximately 60 var genes demethylases (HKDMs) and HKMTs in controlling the that encode variant copies of the surface antigen, Pf transcription of nearly all var genes has also been erythrocyte membrane protein 1 (PfEMP1). The ability demonstrated. Knockout of the Pf hkmt gene encoding to switch expression from one var gene to another enables SET2/SETvs (vs, variant-silencing) resulted in reduced the invading parasite to alternate between phenotypes presence of the repressive H3K36me3 mark at the TSSs of variable cytoadherent and immunogenic properties and intronic promoters of all var gene subtypes (Fig. 3c). [76–78]. PfEMP1 proteins are expressed at parasite- Loss of this SETvs-dependent histone modification re- induced knobs on the infected erythrocyte surface, sulted in the loss of monoallelic var gene expression and which are electron-dense features comprising many expression of the entire var repertoire [98]. Furthermore, parasite proteins anchored to the erythrocyte cytoskel- SETvs can directly interact with the C-terminal domain eton. Failure to present PfEMP1 in such knob structures of RNA II, with SETvs disruption resulting greatly reduces the ability of the infected erythrocyte to in a loss of binding to RNA polymerase II and var gene bind to its specific host receptor [79]. switching [99]. Pf var gene regulation is complex and includes Pf upsA-type var gene expression is also regulated by mechanisms of gene regulation such as chromosomal PfRNaseII, a chromatin-associated exoribonuclease. An organization and subnuclear compartmentalization inverse relationship exists between transcript levels of [80, 81], endogenous var gene clustering and var pro- PfRNaseII and upsA-type var genes, with an increase in moter–intron pairing [82, 83], transcriptional gene silen- the latter corresponding to incidences of severe malaria cing via exoribonuclease-mediated RNA degradation [84], in infected patients [84]. PfRNaseII is proposed to histone variant exchange at var promoters [85, 86], the ef- control upsA-type var gene transcription by marking fect of trans antisense long non-coding RNAs (lncRNAs) TSSs and intronic promoter regions, degrading potential [87], and the presence or absence of histone modifications full-length transcripts to produce short-lived cryptic and their associated histone-modifying enzymes [27, 29, RNA molecules that are then further degraded by the 40, 87–92] (Fig. 3c). Interest in delineating these mecha- exosome immediately upon expression (Fig. 3c). Disrup- nisms has continued, and even grown, as more research in tion of the pfrnaseII gene resulted in loss of this degrad- the post-genomic area has highlighted the important dif- ation and the generation of full-length upsA var gene ferential role of the 5′ upstream promoter families into transcripts and intron-derived antisense lncRNA. These which the var genes can be subdivided into five classes data illustrate the relationship between PfRNaseII and (upsA to upsE), which correlate closely with the severity of the control of monoallelic var gene transcription and malaria infection in the human host [93–98]. Pf var gene suggest a correlation between lncRNA and var gene promoters are also essential components of the gene silen- activation in Pf [84]. The role of lncRNAs in Pf var gene cing mechanism and monoallelic expression. The upsC activation was again investigated in a study by Amit- var promoter in particular is necessary to maintain Avraham and colleagues [87], which demonstrated dose- chromosome-internal var genes in their silenced state and dependent transcriptional activation of var genes by recently has been proposed to do so through the inter- overexpression of their individual antisense lncRNA action of cis-acting MEE2-like sequence motifs and transcripts. Disruption of antisense lncRNA expression MEE2-interacting factors to reinforce var gene transcrip- by peptic nucleic acids resulted in downregulation of tional repression [75, 83]. active var gene transcripts and induced var gene switch- Monoallelic var gene transcription is also associated ing. The exact mechanism by which antisense lncRNAs with the presence of H3K9me3 repressive marks at silent act to promote the active transcription of a var gene is var gene loci (Fig. 3c). This histone modification is unknown. It has been postulated that antisense var predicted but not proven to be imposed by the HKMT transcripts may recruit chromatin-modifying enzymes that PfSET3 and is associated with perinuclear repressive in turn would affect gene accessibility for the Pf transcrip- centers and the binding of PfHP1, stimulating tional machinery. Antisense var gene lncRNAs would also Kirchner et al. Genome Medicine (2016) 8:92 Page 10 of 17

contain a complementary sequence to var gene intronic surface protein PfS47 (found on the surface of the ooki- insulator-like pairing elements that bind specific nuclear- nete as it penetrates the mosquito midgut), that appears binding proteins, therefore blocking the silencing activity to interact with and suppress the vector innate immune of pairing elements by hybridization [87, 100]. system [108]. PfS47 is thought to suppress signaling The Plasmodium helical interspersed subtelomeric pro- through the c-Jun N-terminal kinase (JNK) pathway that tein (PHIST) family of genes, which is unique to Pf, has is critical to an effective immune response [109]. WGS also been implicated in the regulation of immune evasion demonstrated that PfS47 has a distinct population struc- as a result of its ability to bind to the intracellular acidic ture linked to global distribution. PfS47 is rapidly evolv- terminal segment of PfEMP1. Conditional knockdown of ing and selected to achieve JNK suppression in diverse the essential PHIST protein PfE1605w reduced the cap- mosquito species, which becomes a key step in the adap- ability of the infected host erythrocyte to adhere to tation of Pf to transmission in different vectors, thereby the CD36 endothelial receptor, an important virulence contributing to its broad global distribution [110]. feature of Pf. This study highlighted the importance not only of var genes and their controlled expression Artemisinin resistance but also of other genes that are associated with an- The goals of MalariaGEN characterize a new approach choring PfEMP1 to the erythrocyte surface and creat- to understanding parasite population biology. Through ing the Plasmodium cytoadherence complex [101]. the generation and, these days more critically, the man- The list of regulatory mechanisms underlying var gene agement and analysis of the colossal datasets that result monoallelic expression is vast and much more may still from WGS of large numbers of samples, a well- be discovered in this area. However, immune evasion in organized study can draw meaningful conclusions. This the Plasmodium genus is not confined to Pf or var gene was applied to perhaps the most serious threat to mal- regulation. Indeed, var gene expression is exclusive to Pf, aria control that has emerged in recent years—resistance with much still to be gleaned in the areas of immune to ART. Using these datasets in meta-analyses with clin- evasion in human malaria parasites such as P. vivax, P. ical data describing the individual WGS-sequenced sam- knowlesi, Plasmodium ovale and Plasmodium malariae ples and outcomes of ART treatment allowed a path to [13, 102–105]. In addition, PfEMP1 is just one of a be charted that associated SNPs with treatment features number of variant surface antigens (VSAs) known to be (such as delayed clearance) [111] and identified candi- expressed at the host erythrocyte surface upon infection date genes [112]: in both studies a region of chromo- with Pf, although it is the best characterized. Pf-infected some 13 was implicated (Fig. 3d). The precise gene erythrocytes also express VSAs of the multi-copy gene encoding the protein KELCH13 was identified by a com- families of the proteins repetitive interspersed family bination of “old-fashioned” selection of drug-resistant (RIFIN), subtelomeric variable open reading frame parasites in the laboratory followed by WGS and com- (STEVOR), and Pf Maurer’s cleft 2 transmembrane parative genomics of the sensitive parental parasites and (PfMC-2TM) [106]. The roles of these protein families in the progeny, as well as WGS of ART-resistant field iso- antigenic variation and pathology are generally poorly de- lates [113, 114]. The role of the kelch13 mutations in fined but are being elucidated; for example, RIFINs are ART resistance was proven by direct genome engineer- implicated in the severity of Pf malaria in African children ing of kelch13 to generate resistant parasites [115, 116]. with blood group A. This tendency toward increased mal- kelch13 SNPs have been used to map the alarmingly aria pathogenicity is a result of their expression at the sur- rapid spread of resistance throughout Southeast Asia face of infected host erythrocytes, from which they bind [116] and it is clear that there is already significant but uninfected erythrocytes (preferentially, blood group A) to distinct kelch13 heterogeneity in African Pf strains, form rosette structures and mediate binding to the host although there is no evidence of ART resistance microvasculature [107]. Thus, the combined roles of HP1 [117–121]. However, in-depth analysis of Southeast and HDA2 in regulating single var gene expression and Asian ART-resistant parasite genomes [122] revealed that the transcriptional regulator ApiAP2-G suggests that both a complex array of background mutations (Fig. 3d) in a processes share epigenetic regulatory mechanisms and variety of genes (encoding ferredoxin (FD), apicoplast that Plasmodium immune evasion and transmission to ribosomal protein S10 (ARPS10), multidrug resistance new hosts are inextricably linked [27, 29]. protein 2 (MDR2), and Pf chloroquine resistance trans- Immune evasion is not restricted to blood-stage Plas- porter (CRT)) that are not yet described in African modium; when the parasite passes through the mosquito parasites would explain why ART resistance is not (yet) a it must also combat a sophisticated innate immune sys- threat to the use of ART in that continent [121]. tem that is very effective in reducing the parasite burden A further puzzle was the large number of independent experienced by the vector. A forward genetic screen and SNPs that seemed capable of mediating ART resistance— WGS was used to identify the key parasite factor, the typically drug resistance is generated by one or a small Kirchner et al. Genome Medicine (2016) 8:92 Page 11 of 17

number of SNPs focused on either altering the target investigations. A metabolomics-based approach also has binding site for the drug or preventing drug access to a the advantages that parasite lines resistant to the drug binding site buried in the target structure. KELCH pro- need not be generated and that the activity of pleiotropi- teins are propeller proteins with an iterated structural cally acting drugs (such as ART) are directly observed motif that serves as a platform for the assembly of multi- rather than imputed from genomes of resistant parasites. protein complexes. In addition, KELCH13 has a BTB/ POZ domain that might be involved in homodimerization, Vaccines E3 ligase binding, and transcriptional repression Post-genomic approaches have also identified promising (reviewed in [123]). It has been suggested that ART- new Pf vaccine candidates. For example, Pf reticulocyte- resistance-associated kelch13 SNPsmightcauseadegree binding protein homologue 5 (RH5) binds to the human of reduced binding of Pf phosphatidylinositol-3-kinase red cell surface receptor protein basigin, an interaction (PI3K), which in turn results in its reduced ubiquitination that is essential for erythrocyte invasion by Pf [134]. Re- and consequent degradation of PI3K (Fig. 3d). Elevated cent WGS studies have shown that both the host and levels of PI3K generate increased amounts of its lipid parasite proteins are highly conserved, that antibodies to product phosphatidylinositol-3-phosphate (PI3P), which RH5 block merozoite invasion of erythrocytes [135, 136], then changes the physiological state of the parasite cell and that basigin itself is druggable by recombinant anti- through signaling in as yet unknown pathways [124] but bodies [137]. Although RH5–basigin interaction offers through a mechanism predicated on the proposed abun- great promise, the challenges for vaccine development dance of PI3P in the lumen of the endoplasmic reticulum remain considerable and many promising candidates and its proposed role in protein export beyond the para- have fallen or will fall by the wayside due to an inability site vacuole within the host cell [125]. However, aspects of to formulate them to deliver effective vaccination, this view have been challenged [126] and further studies massive candidate gene sequence variability, and func- are clearly required to resolve the possible role of PI3K tional non-essentiality of the candidate. WGS will help signaling in ART resistance. It will be of interest to see if identify non- or minimally variant candidates and should PI3K signaling impacts upon the unfolded protein re- prove useful in monitoring the effect of vaccination and sponse implicated in ART resistance using population the analysis of “breakthrough” parasites (those developing transcriptomics [127]. The WGS data and two in vaccinated individuals), as described in the next section. studies [128, 129] that demonstrate the wide variety of Effective subunit vaccines will be an invaluable additional proteins from different cellular compartments of the tar- approach to vaccination, supplementing other approaches get parasite that interact with activated ART together sug- such as the use of the promising but technologically chal- gest that ART resistance is a pleiotropic phenomenon lenging attenuated whole parasite, for example, sporozoite [123]. Therefore, other interrogations, such as metabolo- vaccine [138]. mics (see next section), might also be needed to gain func- tional insights into ART’smodeofaction. Surveillance The identification of genome signatures of resistance Translational implications for malaria control through WGS in the laboratory and increasingly through Antimalarials large-scale genomic epidemiology provides a powerful tool WGS has been instrumental in identifying the cellular to monitor the emergence of resistance in Plasmodium target of novel Pf antimalarials as part of the drug dis- populations under selective pressure due to administration covery pipeline and in following the in vitro selection of of both drugs and vaccines. In the case of drugs whose resistant parasite lines and validation of observed gen- targets have been identified in the laboratory, specific, omic changes by reverse genetics as described for ART simple PCR-based assays can be devised. WGS of field above. This approach has proved highly successful for parasites under drug pressure is still desirable, however, as spiralindolines [130], resulting in the identification of alternative resistance mechanisms might emerge that the target of NITD609 (also known as KAE609 or cipar- would be missed by targeted assays and, with sufficient gamin) as the P-type ATPase PfATPase4. Furthermore, depth of sampling, new signatures of resistance would be the translation elongation factor eEF2 has been identi- identifiable from the sequence data. Similar surveillance of fied as the target of the 2,6-disubstituted quinoline-4- parasites that emerge post-vaccination might also be in- carboxamide scaffold derivative DDD107498 [131]. WGS formative. An important analysis of the clinical trial of the is not the only post-genome approach that is useful for RTS,S/AS01 malaria vaccine compared the strain-specific determining modes of drug action; metabolomics has a sequence of the gene encoding the circumsporozoite (CS) similar potential for analyzing the metabolic changes protein that comprised the vaccine with the CS gene se- produced in response to drug exposure and has been quences of strains in the infections actually encountered utilized in antibiotic [132] and anti-protozoan drug [133] by immunized individuals (between 5 and 17 months of Kirchner et al. Genome Medicine (2016) 8:92 Page 12 of 17

age) [139]. This study demonstrated that homologous pro- resistance to malaria. Also, new concepts of combatting tection was greater than protection against heterologous malaria via engineered mosquito populations have been strains and that a cause of failure to protect was simply introduced through the advent of novel genome-editing that the CS protein carried by the infecting parasites did approaches such as CRISPR-Cas9. not match that of the vaccine and so perhaps a protective We can anticipate that WGS will continue to improve effect was less likely [139]. Therefore, WGS has the power in terms of both cost and quality, making sequencing of to guide vaccine design based upon the outcomes of trials. every desirable Pf isolate feasible. This would enable more detailed studies of population structure and dy- Gene editing namics, allowing the tracking of gene flow and genotype A new era of genetic engineering has dawned with the success that might even resolve at the village level and, discovery and development of the bacterial guide RNA further, potentially almost in real time. However, this will template-targeted clustered regularly interspaced short only happen if data storage, access, and computing tech- palindromic repeats (CRISPR)-Cas9 recombinases as nologies keep pace. Where Pf WGS studies have gone P. tools for the accurate editing of genomes. The technol- vivax research will follow and recent studies have re- ogy has been successfully adapted to many species, in- vealed signatures of drug selection superimposed upon a cluding Plasmodium [140], Anopheles [141, 142], and far more complex (global, regional, and even within a humans (discussed in [143]). Currently, applications of single infection) population structure than Pf [148, 149]. CRISPR-Cas9 to Plasmodium manipulation are re- Single-cell RNA sequencing will significantly improve stricted to reverse genetic investigations of gene func- our understanding of antigenic variation and variant and tion. However, with the concepts of whole (pre- sex-specific gene expression. erythrocytic) parasite vaccines [144, 145], CRISPR-Cas9 More immediately, an important need is for surveil- offers an obvious route towards the generation of an lance, particularly in Africa, to look for kelch13 muta- immunogenic, non-pathogenic parasite that might be tions and genotypes associated with ART resistance and suitably safe to administer to humans as a vaccination a pan-African network is in place to monitor for this strategy. Clearly, the engineering of human genomes at and collect samples [150]. Genomics will continue to be any stage of gestation is fraught with ethical consider- used in novel ways as well, for example, in studies of the ations [146] and it is inconceivable that this will be ap- outcomes of human interventions such as drug treat- plied to the improvement of human resistance to ment and vaccination. malaria in the foreseeable future. Conversely, although New fields of endeavor are also emerging that will cer- subject to similar ethical and ecological debate, signifi- tainly prove fruitful in the years to come. Lipidomics is a cant conceptual advances towards the generation of nascent discipline that will no doubt reveal insights into CRISPR-Cas9-engineered Anopheles mosquitoes have membrane composition and organization [151] and been quickly achieved. Through harnessing the concept might also open avenues to therapy. PTMs such as pal- of gene drive, two independent teams have reported the mitoylation give proteins the means to conditionally generation of either engineered Anopheles stephensi (a interact with membranes and Plasmodium makes exten- majorIndianvectorofmalaria)thatisresistanttomal- sive use of protein palmitoylation that should influence a aria [141] or sterile female Ag [142]. Again, owing to range of important parasite biological activities, such as ecological considerations, it is unlikely that such engi- cytoadherence and drug resistance [152]. neered mosquitoes, although clearly feasible, will be re- Although the power of genomics approaches is quite leased into the wild any time soon [147]. clear, direct biological investigations are frequently re- quired to confirm or refute the findings that genomics Conclusions and future directions might imply. The numerous examples given here indicate Despite the progress summarized here, the fundamental that although genomic analyses often generate associa- requirements of malaria research in any era remain the tions and degrees of confidence about their conclusions, same; namely, new drugs to replace those that become in- unequivocal confirmation is provided by genetic engineer- effective, vaccines that work, and the means to administer ing (of parasites and their vectors at least). Genetic screens them effectively. Genomics, post-genomic technologies, are powerful, often unbiased approaches to discover gene and associated computing developments have revolution- function. The recent development of the PlasmoGEM ized investigations into the biology of the malaria parasite resource coupled with high-efficiency transfection and and the search for therapeutics or intervention measures. barcoded vectors permits genome-scale reverse genetics Significant progress has been made on many fronts, in- screens to be deployed that will undoubtedly reveal infor- cluding candidate drug and vaccine discovery, parasite mation about parasite-specific genes and Plasmodium drug resistance mechanisms, host–parasite–vector inter- biology [153]. Finally, many of the genes encoded by para- actions and parasite biology, and mechanisms of human site, host, and vector genomes have unknown functions, Kirchner et al. Genome Medicine (2016) 8:92 Page 13 of 17

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The origins of malaria: there are more things in – CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; heaven and earth. Parasitology. 2015;142 Suppl 1:S16 25. DD, Destabilization Domain; G6PD, Glucose-6-phosphate Dehydrogenase; 15. Liu W, Sundararaman SA, Loy DE, Learn GH, Li Y, Plenderleith LJ, et al. GWAS, Genome-wide Association Study; IDC, Intra-erythrocytic Development Multigenomic delineation of Plasmodium species of the Laverania subgenus Cycle; K13, Kelch13; MalariaGEN, Malaria Genomic Epidemiology Network; infecting wild-living chimpanzees and gorillas. Genome Biol Evol. – miRNA, MicroRNA; NGS, Next-generation Sequencing; ORF, Open Reading Frame; 2016;8:1929 39. Pb, Plasmodium Berghei; Pf, Plasmodium Falciparum; PHIST, Plasmodium Helical 16. Mourier T, Carret C, Kyes S, Christodoulou Z, Gardner PP, Jeffares DC, et al. Interspersed Subtelomeric Protein Family; PI3K, Phosphatidylinositol-3-kinase; Genome-wide discovery and verification of novel structured RNAs in – PI3P, Phosphatidylinositol-3-phosphate; PTM, Post-translational Modification; Plasmodium falciparum. Genome Res. 2008;18:281 92. RBC, Red Blood Cell; rDNA, Ribosomal DNA; RIFIN, Repetitive Interspersed 17. Broadbent KM, Broadbent JC, Ribacke U, Wirth D, Rinn JL, Sabeti PC. Family; SNP, Single-nucleotide Polymorphism; TF, Transcription Factor; Strand-specific RNA sequencing in Plasmodium falciparum malaria identifies TSS, Transcription Start Site; UPR, Unfolded Protein Response; VSAs, Variant developmentally regulated long non-coding RNA and circular RNA. BMC Surface Antigens; WGS, Whole-genome Sequencing; ZFN, Zinc Finger Nuclease Genomics. 2015;16:454. 18. Siegel TN, Hon CC, Zhang Q, Lopez-Rubio JJ, Scheidig-Benatar C, Acknowledgements Martins RM, et al. Strand-specific RNA-Seq reveals widespread and SK is a Marie Skłodowska-Curie Fellow (reference 657592). BJP is funded by developmentally regulated transcription of natural antisense transcripts the Wellcome Trust (reference 102463/Z/13/Z). APW is funded by the in Plasmodium falciparum. BMC Genomics. 2014;15:150. Wellcome Trust (reference 083811/Z/07/Z). 19. Manske M, Miotto O, Campino S, Auburn S, Almagro-Garcia J, Maslen G, et al. Analysis of Plasmodium falciparum diversity in natural infections by – Authors’ contributions deep sequencing. Nature. 2012;487:375 9. All authors wrote and proofed the entire manuscript and then read and 20. Bonasio R, Tu S, Reinberg D. Molecular signals of epigenetic states. Science. approved the final manuscript. APW managed the writing and organized the 2010;330:612–6. article. SK produced all figures and tables after mutual discussion and 21. Cui L, Miao J. 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